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PubMed2005

Naked DNA for liver gene transfer.

The majority of acquired and inherited genetic disorders, including most inborn errors of metabolism, are manifested in the liver. Therefore, it is hardly any surprise to see a large number of Medline reports describing gene therapy efforts in preclinical settings directed toward this organ (Inoue et al., 2004; Oka and Chen, 2004). Of late, non-viral vectors have garnered a lot of attention from the biomedical research community engaged in liver gene therapy (Gupta et al., 2004). However, the first initiative toward gene transfer to the liver using a non-viral approach was taken by Hickman et al. (1994), who applied the technique of naked DNA injection pioneered by Wolff (1990) for skeletal muscle. Direct injection of naked DNA resulted in low, variable and localized gene expression in the rat liver. Consequently, several developments reported in the literature since then aimed to improve hepatic gene expression by employing both surgical and nonsurgical methods. These developments include the exploitation of the unique vasculature of liver as well as the use of electric and mechanical force as an adjunct to the systemic administration of the naked plasmid gene. This chapter focuses on these developments reported from various laboratories, including ours. In addition, the underlying mechanism responsible for the dramatic increase in gene expression using these latest approaches for non-viral gene transfer to the liver is also discussed.

Animals↗

PubMed1998

Evaluation of gene therapy for citrullinaemia using murine and bovine models.

Citrullinaemia is an autosomal recessive disorder caused by the deficiency of argininosuccinate synthase. The deficiency of this enzyme results in an interruption in the urea cycle and the inability to dispose of excess ammonia derived from the metabolism of protein. The only treatment for this disorder has been dietary restriction of protein and supplementation with medications allowing for alternative excretion of excess nitrogen. Gene therapy offers the possibility of a long-term cure for disorders like citrullinaemia by expressing the deficient gene in the target organ. We have explored the use of adenoviral vectors as a treatment modality for citrullinaemia in two animal models, a naturally occurring bovine model and a murine model created by molecular mutagenesis. Mice treated with adenoviral vectors expressing argininosuccinate synthase lived significantly longer than untreated animals (11 days vs 1 day; however, the animals did not exhibit normal weight gain during the experiment, indicating that the therapeutic effectiveness of the transducing virus was suboptimal. It is speculated that part of the failure to observe better clinical outcome might be due to the deficiency of arginine. In the bovine model, the use of adenoviral vectors did not result in any change in the clinical condition of the animals or in the level of plasma ammonia. However, the use of 15N isotopic ammonia allowed us to assess the flux of nitrogen through the urea cycle during the experiment. These studies revealed a significant increase in the flux through the urea cycle following administration of adenoviral vectors expressing argininosuccinate synthase. We conclude that the use of adenoviral vectors in the treatment of citrullinaemia is a viable approach to therapy but that it will be necessary to increase the level of transduction and to increase the level of enzyme produced from the recombinant viral vector. Future experiments will be designed to address these issues.

Adenoviruses, Human↗

PubMed2001

Receptor-mediated gene targeting to tissues in vivo following intravenous administration of pegylated immunoliposomes.

PURPOSE: Gene therapy has been limited by the immunogenicity of viral vectors, by the inefficiency of cationic liposomes, and by the rapid degradation in vivo following the injection of naked DNA. The present work describes a new approach that enables the non-invasive, non-viral gene therapy of the brain and peripheral organs following an intravenous injection. METHODS: The plasmid DNA encoding beta-galactosidase is packaged in the interior of neutral liposomes, which are stabilized for in vivo use by surface conjugation with polyethyleglycol (PEG). The tips of about 1% of the PEG strands are attached to a targeting monoclonal antibody (MAb), which acts as a "molecular Trojan Horse" to ferry the liposome carrying the gene across the biological barriers of the brain and other organs. The MAb targets the transferrin receptor, which is enriched at both the blood-brain barrier (BBB), and in peripheral tissues, such as liver and spleen. RESULTS: Expression of the exogenous gene in brain, liver, and spleen was demonstrated with beta-galactosidase histochemistry, which showed persistence of gene expression for at least 6 days after a single intravenous injection of the pegylated immunoliposomes. The persistence of the transgene was confirmed by Southern blot analysis. CONCLUSIONS: Widespread expression of an exogenous gene in brain and peripheral tissues is induced with a single intravenous administration of plasmid DNA packaged in the interior of pegylated immunoliposomes. The liposomes are formulated to target specific receptor systems that enable receptor-mediated endocytosis of the complex into cells in vivo. This approach allows for non-invasive, non-viral gene therapy of the brain.

Animals↗

PubMed2005

Baculovirus as versatile vectors for protein expression in insect and mammalian cells.

Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.

Animals↗

PubMed2006

Therapeutic vaccination against HIV: current progress and future possibilities.

Although effective in reducing mortality, current antiretroviral therapy for HIV infection involves complex and expensive drug regimens that are toxic and difficult to take. Eradication of HIV reservoirs is not possible with existing therapies. The concept of therapeutic vaccination has been investigated to increase the potency and breadth of anti-HIV immune responses in order to delay or reduce antiretroviral therapy use. A variety of approaches targeted to both cell- and antibody-mediated immunity have been developed, including whole inactivated HIV-1, protein subunits and synthetic peptides, DNA vaccines and a number of viral vectors expressing HIV-1. These investigations have occurred in the absence of a clear understanding of disease pathogenesis or the correlates of protective immunity. At this time, there is no licensed therapeutic vaccine for any viral disease, including HIV; however, this review will consider recent progress in the field and summarize the challenges faced in the development of a therapeutic HIV vaccine.

AIDS Vaccines↗

PubMed2005

Adeno-associated virus inverted terminal repeats improve neuronal transgene expression mediated by baculoviral vectors in rat brain.

Baculoviral vectors can transduce neurons in the CNS but mediate only transient expression of transgenes. We have developed a new baculoviral vector in which the inverted terminal repeats (ITRs) of adeno-associated virus are used to flank a luciferase reporter gene cassette harboring a neuron-specific promoter. When tested in rat brain, the new viral vector was able to provide transgene expression for at least 90 days. Immunohistological analysis demonstrated that ITR flanking did not affect the cellular preference of the neuronal promoter in the context of baculovirus. These findings establish an effective way to engineer baculoviral vectors in order to achieve sustained expression of a functional gene for gene therapy for neurodegenerative disorders and physiological studies of neurons.

Animals↗

PubMed2003

[Improvements of lentiviral vector and its application in gene therapy of hematological diseases].

As lentiviral vector holds the characteristics of higher transfection to non-dividing cells, larger capacity of transfer gene fragments, long-term expression of therapeutic gene and lower rate of immunological response, therefore it becomes potential viral vector in gene therapy. Improvements of lentiviral vector, human immunodeficiency virus type-I as example, and its application in gene transfer for gene therapy of hematological diseases are emphasized in this review.

Fanconi Anemia↗

PubMed1998

Genetically engineered antibodies in gene transfer and gene therapy.

Our ability to produce and engineer human monoclonal antibodies provides a basis for the development of novel therapeutical strategies against a variety of diseases. These strategies not only include improved passive immunotherapy but also more sophisticated antibody-based gene therapies involving gene transfer approaches. Four of the major applications of antibody gene engineering in the field of gene therapy are reviewed here. These are (1) the redefinition of viral vector tropism of infection for better transduction of cells of therapeutical interest, (2) the grafting of new cell recognition activities to effector cells of the immune system to kill cancer and pathogen-infected cells, (3) the inhibition of cellular and viral functions through intracellular expression of antibody-derived molecules, and (4) the systemic delivery of therapeutic monoclonal antibodies by non-B cells in living organisms.

Antibodies, Monoclonal↗

PubMed2005

Tumor-specific intravenous gene delivery using oncolytic adenoviruses.

In this report, we describe a vector system that specifically delivers transgene products to tumors following intravenous (i.v.) administration. The Escherichia coli cytosine deaminase (CD) gene was placed in the E3B region of the tumor-selective, replication-competent adenovirus ONYX-411, under the control of endogenous viral late gene regulatory elements. Thus, CD expression was directly coupled to the tumor-selective replication of the viral vector. In vitro, CD was expressed efficiently in various human cancer cell lines tested but not in cultured normal human cells, including human hepatocytes. Following i.v. administration into nude mice carrying human tumor xenografts, robust CD activity was detected only in tumors but not in liver or other normal tissues. Levels of CD activity in the tumors increased progressively following i.v. virus administration, correlating closely with virus replication in vivo. Subsequent administration of 5-fluorocytosine (5-FC) demonstrated a trend to improve the antitumor efficacy of these viruses in a mouse xenograft model, presumably due to the intratumoral conversion of 5-FC to the chemotherapeutic drug 5-fluorouracil. We show that the combination of a highly selective oncolytic virus, ONYX-411, with the strategic use of the viral E3B region for transgene insertion provides a powerful platform that allows for tumor-specific, persistent and robust transgene expression after i.v. administration. This technology provides an opportunity to enhance greatly both safety and efficacy of cancer gene therapy.

Adenoviridae↗

PubMed2002

Gene transfer into the central nervous system in vivo using a recombinanat lentivirus vector.

Gene transfer vectors derived from human immunodeficiency virus (HIV-1) efficiently transduce nondividing cells and may provide for the delivery of their gene products to discrete regions of the brain. We investigated whether stable gene transduction can be achieved in cells of the central nervous system (CNS) in vivo by a potent lentivirus vector. The herpes simplex virus type 1 protein VP22 has been known to facilitate intercellular protein transport and thereby provides an opportunity to increase the effectiveness of therapeutic genes by enhancing the delivery of their protein products. We developed a lentiviral vector construct expressing enhanced green fluorescent protein (EGFP) fused at its N-terminus to the herpes simplex virus VP22. In order to determine expression of the fusion protein in specific cells such as neurons in the CNS, a neuron-specific promoter was also placed into the construct. The viral vectors were injected directly into the striatum and hippocampus of mouse brains. We found that the lentivirus vector efficiently and stably transduced nondividing cells in the CNS with transgene expression for over 3 months. We also show that the delivery of VP22-EGFP fusion protein encoded by the lentivirus was effectively transported between neuronal cells via axons in vivo. Doubly labeled experiments revealed that our lentiviral vector is capable of delivering gene products to neurons and astrocytes in CNS. The data also demonstrate that up to 90% of the CNS cells transduced by our lentiviral vector under the control of the neuronal promoter are neurons.

Animals↗

PubMed2005

Viral stop-and-go along microtubules: taking a ride with dynein and kinesins.

Incoming viral particles move from the cell surface to sites of viral transcription and replication. By contrast, during assembly and egress, subviral nucleoprotein complexes and virions travel back to the plasma membrane. Because diffusion of large molecules is severely restricted in the cytoplasm, viruses use ATP-hydrolyzing molecular motors of the host for propelling along the microtubules, which are the intracellular highways. Recent studies have revealed that, besides travelling inside endocytic or exocytic vesicles, viral proteins interact directly with dynein or kinesin motors. Understanding the molecular mechanisms of cytoplasmic viral transport will aid in the construction of viral vectors for human gene therapy and the search for new antiviral targets.

Dyneins↗

PubMed2005

Intelligent polymers as nonviral vectors.

The successful gene therapy largely depends on the vector type that allows a selective and efficient gene delivery to target cells with minimal toxicity. Nonviral vectors are much safer and cheaper, can be produced easily in large quantities, and have higher genetic material carrying capacity. However, they are generally less efficient in delivering DNA and initiating gene expression as compared to viral vectors, particularly when used in vivo. As nonviral vectors, polycations may work well for efficient cell uptake and endosomal escape, because they do form compact and smaller complexes with plasmid DNA and carry amine groups, which give positive charge and buffering ability that allows safe escape from endosome/lysosome. However, this is a disadvantage in the following step, which is releasing the plasmid DNA within the cytosol. In order to initiate transcription and enhance gene expression, the polymer/plasmid complex should dissociate after releasing from endosome safely and effectively. There are also other limitations with some of the polycationic carriers, for example, aggregation, toxicity, etc. Intelligent polymers, also called as 'stimuli responsive polymers', have a great potential as nonviral vectors to obtain site-, timing-, and duration period-specific gene expression, which is already exhibited in recent studies that are briefly summarized here.

Biotechnology↗

PubMed2005

Gene transfer of human acid sphingomyelinase corrects neuropathology and motor deficits in a mouse model of Niemann-Pick type A disease.

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints. In addition, we evaluated the use of the deep cerebellar nuclei (DCN) as a site for injection to facilitate global distribution of the viral vector and enzyme. Seven-week-old ASM knockout mice were injected within the DCN with different AAV serotype vectors encoding human ASM (hASM) and then killed at either 14 or 20 weeks of age. Results showed that AAV1 was superior to serotypes 2, 5, 7, and 8 in its relative ability to express hASM, alleviate storage accumulation, and correct behavioral deficits. Expression of hASM was found not only within the DCN, but also throughout the cerebellum, brainstem, midbrain, and spinal cord. This finding demonstrates that targeting the DCN is an effective approach for achieving widespread enzyme distribution throughout the CNS. Our results support the continued development of AAV based vectors for gene therapy of the CNS manifestations in Niemann-Pick type A disease.

Animals↗

PubMed2002

Lymphangiogenic gene therapy with minimal blood vascular side effects.

Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema. Furthermore, VEGF-C gene transfer to the skin of mice with lymphedema induced a regeneration of the cutaneous lymphatic vessel network. However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema. To avoid these blood vascular side effects of VEGF-C, we constructed a viral vector for a VEGFR-3-specific mutant form of VEGF-C (VEGF-C156S) for lymphedema gene therapy. We demonstrate that VEGF-C156S potently induces lymphangiogenesis in transgenic mouse embryos, and when applied via viral gene transfer, in normal and lymphedema mice. Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses. In particular, in the lymphedema mice functional cutaneous lymphatic vessels of normal caliber and morphology were detected after long-term expression of VEGF-C156S via an adeno associated virus. These results have important implications for the development of gene therapy for human lymphedema.

Adenoviridae↗

PubMed2015

Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

Animals↗

PubMed2000

Multicolumn infusion of gene therapy cells into human brain tumors: technical report.

OBJECTIVE: Effective gene therapy for brain tumors may require saturation of the tumors with tumoricidal doses of the therapeutic gene. Safe, precise, and efficient delivery of gene therapy vectors is required. Most reported cases of and published protocols for gene therapy for brain tumors involve freehand injection of retroviral vector-producing cells (VPCs) into the brain. Major disadvantages of this method include the inaccuracy of hand-guided needle placement and limited control of injection parameters. These factors can result in failure to deliver the viral vectors to specifically targeted sites within the brain, extensive tissue disruption resulting from excessively forceful injection, and reflux of the injectate along the needle tract. METHODS: We describe a novel stereotactic strategy for saturating tumor volumes with tumoricidal doses of gene therapy vectors and a new, more precise method of infusing VPCs. With our new instrument, the multicolumn stereotactic infusion system, needle placement is stereotactically guided and both VPC infusion and needle withdrawal are mechanically controlled. RESULTS: This technique, which has been used effectively for six patients, permits precise deposition of columns of VPCs throughout the targeted tumor volume. CONCLUSION: This technique should facilitate saturation of tumors with tumoricidal doses of gene therapy vectors and should improve the results of gene therapy protocols that rely on intraparenchymal injection for delivery.

Biopsy↗

PubMed1999

A large-scale insertional mutagenesis screen in zebrafish.

It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.

Alleles↗

PubMed2003

Gene transfer as a future therapy for rheumatoid arthritis.

Inhibiting key pathogenic processes within the rheumatoid synovium is a most attractive goal to achieve, and the number of potential intra- and extracellular pathways operative in rheumatoid arthritis (RA) that could be used for a gene therapy strategy is increasing continuously. Gene transfer or gene therapy might also be one of the approaches to solve the problem of long-term expression of therapeutic genes, in order to replace the frequent application of recombinant proteins, in the future. However, at present, gene therapy has not reached a realistic clinical stage, which is mainly due to severe side effects in humans, the complexity of RA pathophysiology and the current state of available gene transfer techniques. On the other hand, novel gene delivery systems are not restricted to vectors or certain types of cells, as mobile cells including macrophages, dendritic cells, lymphocytes and multipotent stem cells can also be used as smart gene transfer vehicles. Moreover, the observation in animal models that application of viral vectors into a joint can exert additional therapeutic effects in nearby joints might also facilitate the transfer from animal to human gene therapy. Future strategies will also examine the potential of novel long-term expression vectors such as lentiviruses and cytomegalovirus (CMV)-based viruses as a basis for future clinical trials in RA.

Animals↗